TWI421256B - Method for producing 5-ene-3-keto or 3,6-diketone of fatty alcohol, method for producing lipid metabolism improving agent, food and animal and animal feed, and analysis method - Google Patents

Description

Method for producing 5-alken-3-one body or 3,6-dione body of aliphatic alcohol, lipid metabolism improving agent, method for producing food and beverage and animal feed, and analysis method

The present invention relates to a 5-alken-3-one body, a 3,6-dione body of a fatty alcohol, a lipid metabolism improving agent, a method for producing a food or drink, and an animal feed, and an analysis method. More specifically, the present invention relates to a method for producing a 5-en-3-one or a 3,6-dione of an aliphatic alcohol which is excellent in physiological activity such as improvement in anti-obesity and improvement in lipid metabolism, and the like. A lipid metabolism improving agent containing the 5-alken-3-one or 3,6-dione of the aliphatic alcohol, a method for producing a food or beverage and an animal feed, and a 5-alkene capable of simultaneously and efficiently analyzing the aliphatic alcohol Analytical method for 3-keto bodies and 3,6-dione bodies.

It is known that cholesterol derivatives have various physiological effects and are now used in various fields such as medicines, foods, and feeds. For example, Patent Document 1 listed below discloses an anti-obesity agent and a lipid metabolism improving agent containing an active ingredient such as 24-alkylxanthen-3-one or 24-alkylcholest-3-one. Further, Patent Document 2 discloses an anti-obesity agent and a lipid metabolism improving agent containing 24-methylcholest-5-en-3-one of a 5-alken-3-one of a fatty alcohol as an active ingredient. Further, the 5-en-3-one or the 3,6-dione is known to have excellent anti-obesity and lipid metabolism improving effects as compared with the 4-en-3-one of the aliphatic alcohol.

As a method for synthesizing a cholesterol derivative, a biological synthesis method for culturing a microorganism or the like in addition to a chemical synthesis method is known. As a biological synthesis method, for example, the cholestyr-4-en-3-one obtained from the cholesterol oxidase of the enzyme, which is based on cholesterol or a derivative thereof, disclosed in Non-Patent Document 1 below. In this method, a 5-en-3-one is obtained from an oxidation reaction of cholesterol or a derivative thereof, and then a 4-en-3-one is rapidly formed via an isomerization reaction. Further, in Non-Patent Document 2 described below, a microorganism having a cholesterol oxidase-producing property (Arthrobacter) is used in the two layers of the aqueous layer/organic solvent to convert from cholesterol to cholest-4-ene- The biological method of 3-ketone.

Non-Patent Document 1: "Biochemistry and Industry" Vol. 57 No. 7 ('99) 464-467 Non-Patent Document 2 : "Enzyme and Microbial Technology" 1996, Vol. 18: 184-189.

However, cholesterol oxidase, the oxidation rate is smaller than the isomerization rate by several times to several tens of times, and the stage of determining the reaction rate is in the oxidation process. Therefore, in the above non-patent document 1, when the biological synthesis method of cholesterol oxidase is used, the isomerization is rapidly carried out to become a 4-en-3-one, and the yield of 5-en-3-ketone is extremely high. Low problem. Further, the above Non-Patent Document 2 also discloses that only a 4-en-3-one body is biologically obtained, and in the biological synthesis method, there is no technical discovery regarding the improvement of the yield of 5-en-3-one. Further, the above-mentioned Non-Patent Documents 1 and 2 do not disclose the biological synthesis of the 3,6-dione body. Here, it is required to develop a method for producing a 5-en-3-one body of a fatty alcohol which is more excellent in productivity than a conventional method, and a method for producing a 3,6-dione body.

The present invention has been made in view of the above-mentioned state of the art, and provides a 5-en-3-one or a 3,6-dione of an aliphatic alcohol which is excellent in physiological activity such as improvement in anti-obesity and improvement in lipid metabolism. Manufacturing method, and a lipid metabolism improving agent containing the 5-alken-3-one or 3,6-dione of the aliphatic alcohol, a method for producing a food or beverage and an animal feed, and simultaneous and effective analysis of the aliphatic alcohol The analysis method of the 5-en-3-one body and the 3,6-dione body is for the purpose.

In order to solve the above problems, the inventors of the present invention have unexpectedly discovered that a two-layer solvent formed of a hydrocarbon-based solvent layer such as an alkane and a water layer is based on cholesterol and its derivatives, and is oxidized by cholesterol or cultured. The microorganism which exhibits cholesterol oxidase can obtain a 5-en-3-one body or a 3,6-dione body than a conventional high-yield aliphatic alcohol, and the present invention has been completed. In addition, when analyzing the 5-en-3-one and 3,6-dione of the aliphatic alcohol, high-performance liquid chromatography (hereinafter referred to as "high-performance" is found from the specific mobile phase and column temperature. The present invention was completed by simultaneously separating the 5-alken-3-one body and the 3,6-dione body of the aliphatic alcohol by the HPLC method.

The present invention is as follows.

(1) A two-layer solution composed of a water layer formed of a cholesterol oxidase-containing liquid and a hydrocarbon-based solvent layer formed of a hydrocarbon-based solvent, containing cholesterol or a derivative thereof, and reacting as a characteristic aliphatic alcohol -en-3-one Or a method for producing a 3,6-dione.

(2) The method for producing a 5-en-3-one or a 3,6-dione of the aliphatic alcohol according to the above (1), wherein the cholesterol oxidase contains a microorganism culture liquid exhibiting cholesterol oxidase activity.

(3) The method for producing a 5-alken-3-one or a 3,6-dione of the aliphatic alcohol according to the above (2), wherein one or more of the microorganisms belong to the genus Arthrobacter, A bacterium belonging to the genus Streptomyces, the genus Brevibacterium, or the genus Rhodococcus, and the genus Bacillus.

(4) A method for producing a lipid metabolism improving agent characterized by comprising a 5-alken-3-one or a 3,6-dione of an aliphatic alcohol obtained by the method according to (1) above.

(5) A method for producing a food or drink characterized by a 5-alken-3-one or a 3,6-dione obtained from the method described in the above (1).

(6) A method for producing an animal feed characterized by a 5-alken-3-one or a 3,6-dione having an aliphatic alcohol obtained by the method described in the above (1).

(7) Mixing acetonitrile and isopropanol at a volume ratio (3 to 5) / (5 to 7) in a mobile phase, and simultaneously separating the quantitative aliphatic alcohol by high performance liquid chromatography (HPLC) The 5-en-3-one and the 3,6-dione are characterized by a 5-en-3-one and a 3,6-dione of the aliphatic alcohol.

(8) A method for analyzing a 5-en-3-one and a 3,6-dione of the aliphatic alcohol according to the above (7), wherein the column temperature in the high performance liquid chromatography (HPLC) method is 10 Up to 30 ° C.

The method for producing a 5-en-3-one or a 3,6-dione of the aliphatic alcohol of the present invention is excellent in physiological effects such as anti-obesity and improvement in lipid metabolism which is higher than a conventional yield by having the above composition. a 5-en-3-one or a 3,6-dione of a fatty alcohol.

Further, the method for producing a lipid metabolism improving agent, a food or drink, and an animal feed of the present invention has the above-described composition, and a lipid metabolism improving agent, a food or beverage, and an animal feed which achieve the above-described excellent effects can be obtained more efficiently than conventionally.

Further, from the analysis method of the 5-en-3-one body and the 3,6-dione body of the aliphatic alcohol of the present invention, it has been found that 5-alken-3-one of the aliphatic alcohol can be simultaneously separated and quantified by a conventionally difficult HPLC method. And 3,6-dione.

[The best form for implementing the invention]

The invention is illustrated as follows.

The "aqueous layer formed of the cholesterol oxidase-containing liquid" constituting the above-mentioned "two-layer solution" of the present invention contains a cholesterol oxidase layer in the aqueous solvent. The type of the aqueous solvent is not particularly limited as long as it has a property of not being mixed with the hydrocarbon-based solvent and separated into two layers. As the aqueous solvent, water is usually used.

The above "cholesterol oxidase" catalyzes a reaction between a molecule of a 3?-hydroxy steroid and one molecule of oxygen, and produces a corresponding enzyme of 3-carbonyl steroid and hydrogen peroxide (EC. 1.1.3.6). The above-mentioned cholesterol oxidase is a natural cholesterol oxidase which is usually produced by a microorganism. However, as long as it has a catalytic activity, the amino acid oxidase may be changed by a genetic engineering method or the like, and an amino acid sequence or a high-order structure may be used.

The cholesterol oxidase-containing solution is usually prepared by directly adding and containing cholesterol oxidase in an aqueous solvent, but may be prepared by adding or culturing a microorganism exhibiting cholesterol oxidase activity to an aqueous solvent. In other words, the cholesterol oxidase-containing solution further contains a microorganism-containing solution showing cholesterol oxidase activity.

The above-mentioned "microorganism" is a microorganism which exhibits cholesterol oxidase activity, and there is no particular limitation on the kind thereof. For example, the above-mentioned "microorganisms" include, for example, Basidiomycetes such as the genus Agaricus or Coriolus versicolor, filamentous fungi such as the genus Aspergillus and the genus Monascus, and belong to the genus Arthrobacter. (Arthrobacter genus, Streptomyces genus, Brevibacterium genus, or Rhodococcus genus, and Bacillus genus bacteria. Specific examples of the microorganism include, for example, Arthrobacter Simplex (alias Nocardioidos Simplex).

The above microorganisms usually use commercially available microorganisms, but others are naturally or by EMS (ethylmethanesulfonate), diethylsulfonate, NTG (N-methyl-N'-nitro-N-Asia A chemical substance such as a nitroguanidine or a nitrosoguanidine, or a microorganism having a bacteriological mutation obtained by a human mutagenesis means such as X-ray, γ-ray or ultraviolet light can be used as long as it exhibits cholesterol oxidase activity. In addition, a gene encoding cholesterol oxidase may be introduced into a microorganism such as coliform by a suitable vector or the like, and subjected to transformation, and a transformed microorganism capable of producing cholesterol oxidase may be used.

The pH of the cholesterol oxidase-containing solution is not particularly limited as long as it does not impair the cholesterol oxidase activity, and can be determined in accordance with the cholesterol oxidase or the type of microorganism to be used. The cholesterol oxidase-containing solution has a pH of usually 5.0 to 9.0, preferably 6.0 to 8.0, more preferably 6.5 to 7.5. The pH of the cholesterol oxidase-containing solution can be adjusted, for example, by adding an alkaline solution such as an aqueous ammonia solution, a potassium hydroxide aqueous solution, a sodium hydroxide aqueous solution, a sodium carbonate aqueous solution or a potassium carbonate aqueous solution, or an acidic solution such as phosphoric acid.

The cholesterol oxidase-containing solution must contain the above-mentioned cholesterol oxidase or the above-mentioned microorganism exhibiting cholesterol oxidase activity, but may contain other components as long as the effects of the present invention are not impaired. For example, it may contain a carbon source, a nitrogen source, an inorganic salt, an amino acid, a vitamin, and the like which are required for growth and development of a microorganism exhibiting the above cholesterol oxidase activity. In addition, it may also contain a coenzyme for the action of the above cholesterol oxidase. Further, a pH adjuster for adjusting the pH of the cholesterol oxidase-containing solution may be contained.

The content of the above-mentioned cholesterol oxidase in the cholesterol oxidase-containing solution and the number of the microorganisms are not particularly limited, and the appropriate content and the number of bacteria can be determined as needed.

The "two-layer solution" constituting the present invention is a solvent having a property of being separated into two layers as long as the above-mentioned "hydrocarbon-based solvent layer formed of a hydrocarbon-based solvent" is not completely mixed with the water layer constituting the two-layer solution. , you can use it. The "hydrocarbon solvent" may be any of an aliphatic hydrocarbon, an alicyclic hydrocarbon, and an aromatic hydrocarbon solvent. Further, one type of these may be used, or two or more types of mixed solvents may be used. The number of carbon atoms in the above-mentioned "hydrocarbon-based solvent" is not particularly limited, but is usually 4 or more carbon atoms, preferably 4 to 12 carbon atoms, and preferably 5 to 8 hydrocarbon-based solvents. Specific examples of the alkane-based solvent include, for example, an alkane such as n-butane, n-hexane or heptane, or a cycloalkane such as cyclohexane. Further, the hydrocarbon-based solvent may contain other substances as long as it does not inhibit the effects of the present invention.

In the present invention, a hydrocarbon-based solvent is added to the aqueous layer formed of the cholesterol oxidase-containing solution to prepare a two-layer solution composed of two layers of the aqueous layer-hydrocarbon-based solvent layer. In addition, when the two-layer solution is prepared, the order of addition of the cholesterol oxidase-containing solution and the hydrocarbon-based solvent is not particularly limited. For example, the cholesterol oxidase-containing solution may be added to the hydrocarbon-based solvent, and conversely, the hydrocarbon-based solvent may be added to the cholesterol oxidase-containing solution. Further, the ratio of the water layer to the hydrocarbon-based solvent layer in the two-layer solution is not particularly limited, and various ranges can be determined as needed. Usually, the ratio (volume ratio) of the aqueous layer to the hydrocarbon-based solvent layer is 1: (0.2: 2), preferably 1: (0.4 - 1.5), and preferably 1: (0.5-1).

In the present invention, the above two-layer solution contains a "lipool or a derivative thereof" which is a matrix, and is reacted by the above cholesterol oxidase. The type of the above-mentioned "fatty alcohol" is not particularly limited, and various derivatives can be used as needed. Examples of the above-mentioned "aliphatic alcohol or a derivative thereof" include phyto-alcohol (a plant-derived aliphatic alcohol and a derivative thereof, such as β-guol, campesterol, stigmasterol, etc.), cholesterol, ergosterol, and wool. Derivatives such as sterol, cholestyl-3-one, cholestyen-3-one, and the like, such as 24-alkyl.

The method of containing the above-mentioned "aliphatic alcohol or its derivative" in the above two-layer solution is not particularly limited. For example, after the above two-layer solution is prepared, "aliphatic alcohol or a derivative thereof" may be added. Or adding a "lipool or a derivative thereof" to the cholesterol oxidase-containing solution or the hydrocarbon-based solvent before the preparation of the two-layer solution, and secondly mixing the cholesterol oxidase-containing solution and the hydrocarbon-based solvent, or preparing the mixture The above two-layer solution contains the above-mentioned "aliphatic alcohol or its derivative".

The above reaction conditions are not particularly limited. For example, the reaction temperature is usually from 25 to 42 ° C, preferably from 28 to 38 ° C. Further, the reaction time is usually from 5 to 72 hours, particularly preferably from 24 to 48 hours. Further, it is also possible to appropriately vibrate or stir in the reaction.

The method for purifying and recovering the 5-en-3-one or the 3,6-dione of the aliphatic alcohol obtained by the present invention is not particularly limited, and may be recovered and purified by an appropriate means as needed. Usually, in the reaction liquid, a hydrocarbon solvent layer containing a 5-alken-3-one or a 3,6-dione of a lipool is separated, and if necessary, a solvent or the like is distilled off to concentrate the active ingredient, followed by freeze-drying, It can be recovered by drying by a suitable method such as spray drying or vacuum drying. Further, in the purification and recovery method, the reaction liquid or the separated hydrocarbon solvent layer may be sterilized by treatment with a sterilizing filter or the like as needed. Further, in the above-mentioned recovery method, when an alcohol such as methanol or ethanol is added to the reaction liquid, the reaction liquid is easily separated from the two layers of the aqueous layer-hydrocarbon solvent layer, and as a result, it is easy to purify and recover the aliphatic alcohol. a 5-en-3-one or a 3,6-dione.

The "5-en-3-one of the aliphatic alcohol" obtained by the present invention is not particularly limited as long as it has a double bond at the 5th position of the aliphatic alcohol skeleton and has a ketone based on the third position. In addition, the "3,6-dione body of a fatty alcohol" obtained by the present invention is not particularly limited as long as it has a ketone based on the third and sixth positions of the aliphatic alcohol skeleton. For example, an appropriate function such as an alkyl group (methyl group, ethyl group, etc.) may be used based on the aliphatic alcohol skeleton or the branch. Further, the number of double bonds is not particularly limited, and may be one or more in the aliphatic alcohol skeleton or the branch, preferably from 1 to 4, more preferably from 1 to 2 other double bonds.

Specific examples of the above "5-en-3-one of the aliphatic alcohol" include, for example, 24-alkyl cholest-5-en-3-one and 24-alkyl cholestyl-5,7-diene. 3-ketone, 24-alkyl cholestyl-5,8-dien-3-one, 24-alkyl cholest-5,9(11)-dien-3-one, 24-alkylcholine -5,22-dien-3-one, 24-alkyl cholestyl-5,7,22-trien-3-one, 24-alkyl cholestyl-5,8,22-triene-3- Ketone, 24-alkylcholine-5,9(11),22-trien-3-one, 24-alkylcholine-5,25(27)-dien-3-one, and the like. In addition, examples of the "alkyl group" in each of the above substances include a methyl group or an ethyl group. Further, as the above-mentioned "3,6-dione of a fatty alcohol", specifically, for example, 4-cholestene-3,6-dione, 4-calyxene-3,6-dione, 24-alkane Base bile-4-ene-3,6-dione and the like.

The present invention can be carried out to obtain a 5-en-3-one body, or can be carried out to obtain a 3,6-dione body. Further, since the present invention can simultaneously obtain both a 5-en-3-one body and a 3,6-dione body, it is also possible to obtain both at the same time.

The method for separating and quantifying the 5-en-3-one body and the 3,6-dione body of the aliphatic alcohol obtained by the present invention is not particularly limited, and may be carried out by various methods as needed. It is generally carried out by the analytical method of the invention as detailed below.

The 5-en-3-one body and the 3,6-dione body of the aliphatic alcohol obtained by the present invention have excellent anti-obesity and lipid metabolism improving effects, etc., compared with the 4-ene-3 ketone body of the aliphatic alcohol. Physiological effects. Therefore, the 5-en-3-one system of the aliphatic alcohol obtained by the present invention can be used for various purposes. For example, an additive for pharmaceuticals, foods and drinks, and an additive for feed can be used as an anti-obesity agent and a lipid metabolism improving agent. As a result, a pharmaceutical product such as an anti-obesity agent and a lipid metabolism improving agent containing a 5-alken-3-one of a fatty alcohol, a health drink or a health food having an anti-obesity and a lipid metabolism improving effect, and a meat quality improvement can be obtained. A feed for livestock or a feed for fish, and a pet food for preventing obesity in animals, and the like.

The analysis method of the 5-en-3-one body and the 3,6-dione body of the aliphatic alcohol of the present invention is obtained by mixing acetonitrile and isopropyl alcohol in a ratio of (3 to 5) / (5 to 7) by volume. The mixed solution is a mobile phase, and is characterized by high performance liquid chromatography (HPLC), which simultaneously separates and quantifies the 5-alken-3-one body and the 3,6-dione body of the aliphatic alcohol.

In the analysis method of the 5-en-3-one body and the 3,6-dione body of the aliphatic alcohol of the present invention, acetonitrile and isopropyl alcohol are used as the mixed liquid. Secondly, the ratio of acetonitrile to isopropyl alcohol is preferably a ratio of (3 to 5) / (5 to 7) on a volume basis, and a ratio of (3.5 - 4.5) / (5.5 - 6.5) is preferred. By determining the relevant range, it is possible to realize a 5-en-3-one body and a 3,6-dione body which are conventionally difficult to simultaneously separate and quantify a fatty alcohol. Further, in the above HPLC method, other conditions are not particularly limited, but the type, length, temperature, mobile phase flow rate, and the like of the column may be determined in various ranges as needed. For example, in terms of separation efficiency and efficiency of action, the length of the column is 20 to 80 cm, preferably 40 to 60 cm. Further, the column temperature is 10 to 30 ° C, preferably 15 to 25 ° C. Further, the flow rate of the mobile phase is preferably 0.1 to 5 ml/min, and particularly preferably 0.1 to 1 ml/min.

[Examples]

Hereinafter, the present invention will be specifically described by way of examples.

An aqueous liquid medium (pH: 7.0 ± 0.1) having the composition shown in the following Table 1 (A) was prepared as a preculture medium. Further, an aqueous liquid medium (pH: 7.0 ± 0.1) having the composition shown in the following Table 1 (B) was prepared as the present culture medium. The commercially available Arthrobacter Simplex using positive bacteria was used as the bacterial system.

The above-mentioned cells of one platinum ear were scraped from activeslant, and the above culture medium was implanted. Next, the culture was shaken for 2 days at 30 ° C under conditions of about 180 rpm. Next, the pre-culture medium was implanted in the above culture medium to make the concentration of the bacteria 2%. Next, the ventilating amount was appropriately adjusted to 1 VVM, pH was 7.0 ± 0.2, temperature was 30 ° C ± 0.3, paddle rotation number was 250 rpm, and the conditions of dissolved oxygen were continued for 2 days. In this case, 0.1% of a phytolitol "PHS-FK" (manufactured by TAMA Biochemical Co., Ltd.) and 0.1% of an emulsifier "O-10V" (manufactured by Kao Corporation) were added as an inducer of cholesterol oxidase.

After the completion of the culture, 100 ml of the culture medium was added with an equal amount of hexane, and a substrate as shown in the following Table 2 was added thereto to have a final concentration of 1%, and the aqueous layer-hydrocarbon solvent layer was adjusted. Two layer solution. Next, after the culture medium was sealed, the mixture was stirred until the upper layer and the lower layer were mixed, and the reaction was continued for 2 days. After the completion of the reaction, ethanol was added twice as much as the above two-layer solution, and the mixture was stirred, and then a little ethanol was gradually added to separate the upper layer (hydrocarbon-based solvent layer) from the lower layer (aqueous layer). After the lower layer was completely removed, the upper layer was passed through a sterilizing filter, and secondly, dried by a vacuum dryer to recover a powdery product. This product was analyzed by HPLC. The results are shown in Table 2 below. Further, as a comparative example, cholesterol was used as a substrate, and the product was analyzed in the same manner as above except that the above hexane was not added, and the product was analyzed. The results are also reported in Table 2 below.

HPLC analysis of the product was carried out by the following method.

Acetonitrile and isopropanol were mixed at a ratio of 4/6 (volume basis), degassed under reduced pressure using an aspirator, and the mobile phase was prepared. Next, the mobile phase of the liquid column is 10 times the amount of the mobile phase, and the column is balanced. The column balance system detects the baseline and completes if the baseline is stable.

Next, the above product was accurately weighed and dissolved in the above mobile phase to form 0.2% (v/v). After dissolution, the filtrate was treated with a filter (0.42 μm) as an HPLC sample. Next, HPLC was carried out under the following conditions.

(HPLC conditions)

Column: "Cadenza CD C-18" (4.6 × 500mm; manufactured by Imtakt) Flow: 0.55ml / min Detector and detection wavelength: "UV detector" 210nm column temperature: 18 ° C sample dissolved solvent: and mobile phase The same injection amount: 20 μl The content of each component in the sample was determined from the obtained peak area and the calibration curve, and the concentration of each component was quantified by the following formula.

Concentration of each component (%, v/v) = content of each component in the sample determined by the calibration curve × 100/40

As is clear from Table 2, when the reaction was carried out in the absence of a hydrocarbon-based solvent hexane layer, as shown in Comparative Example 1, the obtained product was only a 4-ene group, and the 5-ene group and the 3, 6 of the object of the present invention could not be obtained. - Diketone body.

On the other hand, in the two-layer solution composed of the aqueous layer formed of the cholesterol oxidase-containing liquid and the hydrocarbon-based solvent hexane layer, the matrix was added and the reaction was carried out, and Example 1 was different from Comparative Example 1. A 5-olefin can be obtained. Further, when phytolipid was used as a substrate, as shown in Example 2, it was found that a 5-olefin was obtained, and a 3,6-dione body was also obtained.

Further, the present invention is not limited to the specific embodiments described above, and various modifications may be made within the scope of the invention in light of the object and the application.

[industrial use]

The present invention is effective for producing a 5-en-3-one body or a 3,6-dione body of a fatty alcohol having excellent physiological effects such as anti-obesity and lipid metabolism improving action. The present invention is suitable for use in the fields of food and beverage, pharmaceuticals, animal feed, and the like.

Claims (6)

A method for producing a 5-alken-3-one or a 3,6-dione of a fatty alcohol, characterized in that the aqueous layer formed from the cholesterol oxidase-containing solution is at least selected from the group consisting of n-hexane and heptane a two-layer solution comprising a hydrocarbon-based solvent layer formed by a hydrocarbon-based solvent, comprising a phyto-alcohol, and reacting the phytoaleol with the cholesterol oxidase; wherein the cholesterol oxidase-containing solution is formed The aqueous layer is a microbial culture liquid showing cholesterol oxidase, which is a genus of Arthrobacter. A method for producing a 5-en-3-one or a 3,6-dione of a fatty alcohol according to the first aspect of the invention, wherein the cholesterol oxidase-containing solution has a pH of 5.0 to 9.0. A method for producing a 5-en-3-one or a 3,6-dione of a fatty alcohol according to claim 1 or 2, wherein after the end of the reaction, an alcohol is added to the reaction liquid. A method for producing a 5-en-3-one or a 3,6-dione of a fatty alcohol according to the first aspect of the patent application, which is a method for producing a lipid metabolism improving agent. A method for producing a 5-en-3-one or a 3,6-dione of a fatty alcohol according to the first aspect of the invention, which is a method for producing a food or drink. A method for producing a 5-en-3-one or a 3,6-dione of a fatty alcohol according to the first aspect of the patent application, which is a method for producing a feed for animals.